1. Summary of the Invention
The present invention relates to a sustainable chicken cell line infected with Marek's disease virus (MDV). In particular, the present invention relates to a cell line which can be used as a virus vaccine, as a system in which to produce altered MDV strains, and which can be used to determine the affect of various reagents or growth conditions on the cell line over time.
2. Description of Related Art
Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens, characterized by lymphocytic infiltration in visceral organs, muscles, and peripheral nerves. The etiological agent of MD, an avian herpesvirus called Marek's disease virus (MDV), is highly infectious and cell associated (Calnek, B. W., and R. L. Witter, In "Diseases of Poultry: Marek's Disease" (B. W. Calnek, et al., Eds.), pp. 342-385. Iowa State University press, Ames, Iowa (1991)). MDV replicates in a productive restrictive manner in B-lymphocytes and cells growing in tissue culture. Production of fully enveloped virus is restricted to feather follicle epithelium of infected birds (Witter, R. L., et al., J. Natl. Cancer Inst. 49:1121-1130 (1972); Calnek, B. W., et al., Avian Dis. 14:219-233 (1970)). MDV rapidly establishes a latent infection in T-lymphocytes, ultimately leading to malignant transformation and neoplastic disease (Shek, W. R., et al., J. Natl. Cancer Inst. 70:485-491 (1983)). However, the precise relationship between latency and transformation in MDV infected T-lymphocytes is unknown. Akiyama, Y., et al. (Continuous cell culture from lymphoma of Marek's disease. Biken J. 16:177-179 (1973)) first succeeded in establishing a T-lymphoblastoid cell line from MD-infected chickens. Since then, more than 80 cell lines have been produced from MD lymphomas (Akiyama, Y., et al., Two cell lines from lymphomas of Marek's disease. Biken J. 17:105-116 (1974); Powell, P. C., et al., Nature 251:79-80 (1974); Calnek, B. W., et al., Int. J. Cancer 21:100-107 (1978); Payne, L. N., et al., Int. J. Cancer 28:757-766 (1981); Nazerian K., and R. L. Witter, J. Natl. Cancer Inst. 54:435-458 (1975)). Although suitable for some studies, these cell lines are many passages removed from the original event(s) leading to their transformation.
Evidence suggests that viral genomes in MD-lymphoblastoid cell lines are predominately integrated into cellular chromosomes, but episomal forms also exist (Delecluse, H.-J., et al., J. Virol. 67:82-92 (1993)). Analysis of viral transcription in transformed lymphoblastoid cell lines has revealed variable but limited transcriptional activity confined to approximately 20% of the viral genome (Maray, T., et al., Virus Genes 2:49-68 (1988)). MDV-specific transcripts in transformed lymphoblastoid cells are primarily derived from within long and short region terminal repeats (TRL, and TRS respectively) and internal repeats (IRL and IRR respectively). Little transcriptional activity is detected within either the long unique (U.sub.L) or short unique (U.sub.S) regions (Sugaya, K., et al., J. Virol. 64:5773-5782 (1990)). MDV can be rescued from some lymphoblastoid cell lines by co-cultivation with primary or secondary chicken and duck embryo fibroblasts (CEF and DEF, respectively), which support the lytic cycle of MDV in vitro (Schat, K. A., et al., Int. J. Cancer 44:101-109 (1989)). In addition, some lymphoblastoid cell lines will induce MD upon injection into susceptible birds (Akiyama, Y., et al., Continuous cell culture from lymphoma of Marek's disease. Biken J. 16:177-179 (1973); Nazerian, K., et al., Avian Diseases 21:69-76 (1977)).
Since development of live virus Marek's disease vaccines in the late 1970's, losses to Marek's disease have been significantly reduced (Calnek and Witter, In "Diseases of Poultry: Marek's Disease" (B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder, Jr., Eds.), pp. 342-385. Iowa State University press, Ames IA (1991)). The most widely used Marek's disease vaccines are live Turkey herpesvirus (HVT or serotype 3 MDV) or a bivalent mixture of HVT and the apathogenic serotype 2 strain of MDV (MDV-2). The bivalent mixture of HVT and serotype 2 MDV synergistically affords greater protection against Marek's disease, especially in those situations where HVT is not fully effective (Witter, R. L., Protection by attenuated and polyvalent vaccines against highly virulent strain of Marek's disease virus. Avian Path. 11:49-62 (1982); Witter, R. L. and L. F. Lee, Polyvalent Marek's disease vaccines: safety, efficacy and protective synergism in chickens with maternal antibodies. Avian Path. 13:75-92 (1984); Witter, R. L., Principles of Vaccination. In "Marek's Disease: Scientific Basis and Methods of Control" (Payne, L. N., ed.) pp. 203-250. Marinus Nijhoff Pub., Boston, Mass. (1985)). Marek's disease vaccines are the most widely used vaccines in the poultry industry. Current Marek's disease vaccines are either suspensions of infected chicken embryo fibroblasts (CEF) or cell-free virus suspensions made from sonicated CEF infected with vaccine strains of Marek's disease virus (MDV).
Two major difficulties in working with MDV are the strongly cell associated nature of the virus and the lack a sustainable cell culture system amenable to productive (lytic) infections. Primary CEF and DEF are permissive for MDV replication. However, these cultures have a finite life span (approximately 3 weeks), thus necessitating passage of infected primary cells onto an uninfected cell monolayer to propagate MDV and to obtain sufficient quantities of virus with which to work. Such conditions also preclude establishment of one-step growth experiments for effective temporal gene regulation studies. The finite life span of CEF and DEF also make positive selection in mutagenesis studies difficult.
The poultry industry has always recognized the need for continuous avian cell lines that could be used in producing Marek's disease vaccines and simplify development of recombinant MDV vectors for polyvalent vaccines. Although many avian cell lines have been developed (Nazarian, K., An updated list of avian cell lines and transplantable tumors. Avian Path. 12:527-544 (1987)), none of these can substitute for CEF cells in vaccine production. Previous cell lines failed because they were either derived from virally transformed cells or, if derived from chemically transformed cells, the cells produced tumors when inoculated into chickens. Since there are no sustainable cell lines suitable for propagating MDV, the MDV vaccine industry uses primary chicken embryo fibroblasts (CEF) for production of vaccine virus (Churchill, A. E., Production of Vaccines. In "Marek's Disease: Scientific Basis and Methods of Control" (Payne, L. N., ed.), pp. 251-265. Marinus Nijhoff Publishing, Boston, Massachusetts (1985)). Since primary CEFs have a finite life span, they must be prepared every week, increasing costs for producing MDV vaccines. For example, one major United States MDV vaccine producer utilizes 25,000 chick embryos every week. It is estimated that costs associated with purchase and preparation of chick embryos accounts for 40 to 45% of the total cost of MDV vaccine production. A significant reduction in MDV vaccine production costs could be realized if a continuous cell line suitable for vaccine production were established. Requirements for such a cell line are: 1) The cell line can not be virally transformed, 2) Chemically transformed cell lines must be incapable of inducing tumors in vaccinated chickens, and 3) Virus titers produced by such a cell line must be equivalent (or nearly so) to titers obtained through infection of primary cells.
Development of a continuous cell line which would support MDV replication would alleviate many of the difficulties associated with MDV experimentation and vaccine production.